The purpose of this proposal is to use hepatoma cells in culture and/or hepatic tissue from obese, diabetic mice to elucidate the intracellular consequences of insulin binding to its cell surface receptor. The proposal is divided into three complementary studies: 1) A correlation of insulin binding to the intracellular action of insulin as determined by the regulation of the synthesis of the enzyme tyrosine aminotransferase (TAT); 2) Studies of the mechanism of insulin action using its interaction with glucocorticoid hormones and dibutyryl cAMP in regulating the synthesis of TAT and phosphoenolpyruvate carboxykinase (PEPCK), presumably through regulation of the synthesis or translational systems which are capable of synthesizing TAT and PEPCK. We also will attempt to purify these mRNAs so that a cDNA probe can be employed as a more sensitive assay; 3) Use of in vitro systems to identify the intracellular mediator of insulin action with respect to the synthesis of TAT and PEPCK mRNA. This system will be used to search for other small molecules or macromolecules which can regulate the synthesis of these proteins. We also will test for such effects in a system consisting of rat polysomes and in a translational system developed entirely from rat liver. The studies will compare normal tissue with that in which the insulin receptor concentration is down-regulated. The significance of these studies is that they should afford direct evidence of the relationship between insulin receptor concentration and occupancy with specific biochemical effects of insulin. These studies may also provide a means for identifying and isolating the intracellular mediator(s) of insulin action.